类胚体中残留细胞分化全能性维持与外源性白血病抑制分化因子的关系

目的 研究小鼠胚胎干细胞体外类胚体分化后残留未分化细胞的分化能力，探讨其分化全能性维持与外源性白血病抑制分化因子（LIF）的关系。方法 小鼠R1胚胎干细胞体外经20 d的类胚体诱导分化后，胰酶消化为单细胞悬液，在不含LIF的DMEM培养液中贴壁培养。扩增后的细胞用流式细胞仪分析未分化及分化表面标志（CD9、SSEA1和Flk-1）的表达情况。分别于再次类胚体诱导分化前后，RT-PCR检测Oct-4、Nanog、Rex1、FGF5、Nestin、Brachyury、Flk-1和GATA6表达。将扩增后的残留细胞注射至裸鼠皮下，检测畸胎瘤形成能力。Oct-4/GFP 转基因小鼠胚胎干细胞在半固体培养基上作单细胞分化，20 d后统计分化残留率。结果 在不含LIF的DMEM培养液中的残留细胞呈克隆样生长，表达未分化胚胎干细胞标志CD9、SSEA1、Oct-4、Nanog和Rex1。残留细胞体外可再次诱导分化，表达3个胚层的分化标志。残留细胞裸鼠皮下注射6周后形成畸胎瘤。单细胞分化实验表明，常规培养的小鼠胚胎干细胞中约14%的细胞具有分化残留能力。结论 残留胚胎干细胞全能性维持不依赖外源性白血病抑制分化因子；常规培养的胚胎干细胞仅部分具有残留能力。

Relationship between pluripotency maintenance of residual embryonic stem cells and exogenous leukemia inhibitory factor

Objective To investigate the pluripotency of residual undifferentiated embryonic stem (ES) cells from differentiated embryoid bodies (EBs), and explore the relationship between pluripotency maintenance and exogenous leukemia inhibitory factor (LIF). Methods Mouse R1 ES cells were differentiated for 20 days to form EBs. EBs were then trypsinized and re-plated on tissue culture plate in DMEM without LIF. The expression of phenotypes (CD9, SSEA1 and Flk-1）of expanded cells was analysed by flow cytometry. The expression of Oct-4, Nanog, Rex1, FGF5, Nestin, Brachyury, Flk-1 and GATA6 was detected by RT-PCR before and after secondary EB differentiation. The residual cells after expansion were subcutaneously injected into nude mice, and the ability to form teratoma was examined. Single Oct-4/GFP transgenic mouse ES cells were differentiated in semi-solid media to determine the residual rates after 20 days. Results The residual cells grew out to form ES-like colonies in DMEM without LIF, and expressed undifferentiated ES markers such as CD9, SSEA1, Oct-4, Nanog and Rex1. Meanwhile, the residual cells could be redifferentiated to form EBs and express Nestin, Brachyury, Flk-1 and GATA6. Teratoma was formed 6 weeks after subcutaneous injection of residual cells into nude mice. Single cell differentiation of Oct-4-GFP cells showed that about 14% ES cells in primary culture possessed the potential to generate residual undifferentiated cells after long-term differentiation. Conclusion The pluripotency of residual cells from differentiated EBs can be maintained without exogenous LIF. Only a part of the primary cultured ES cells possess the potential to generate residual undifferentiated cells after long-term differentiation.